Receptor subtype and function selective retinoid and rexinoid compounds in combination with immune modulators for cancer immunotherapy

ABSTRACT

Disclosed herein are methods for treating cancer comprising administering at least one immune checkpoint inhibitor and at least one Retinoic Acid Receptor or Retinoid X Receptor active agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/620,411, filed Jun. 12, 2017, which claims the benefit of U.S.Provisional patent application 62/348,646 filed Jun. 10, 2016 and62/406,779 filed Oct. 11, 2016, each of which is hereby incorporated byreference in its entirety.

BACKGROUND

The immune surveillance system provides a host with defense againstforeign antigens, but also limits activity against self-antigens therebypreventing autoimmune disease. Cell surface immune checkpoint moleculesare endogenous regulators of the immune response which limitautoimmunity by enabling co-inhibitory signaling pathways. Immunecheckpoint pathways are important in tumor growth by leading to T-celldepletion and allowing tumors to escape from immune surveillance andenabling unchecked tumor growth. Molecules such as monoclonal antibodies(mAbs) can be designed to target immune checkpoints and such moleculescan antagonize co-inhibitory immune pathways, restore immunesurveillance, and produce an anti-tumor immune response.

Also, the full anti-tumor effects of immunotherapies can be derived onlywhen they are used in combination with other compounds which canovercome resistance that develop to the immunotherapies by modulatingimmune pathways.

SUMMARY

Disclosed herein are compounds for potentiation of immune checkpointtargeted therapies. Compounds which act on retinoic acid receptors (RAR)and retinoid X receptors (RXR) potentiate the activity of immunecheckpoint targeted agents.

Thus, provided herein are methods of treating cancer, the methodscomprising administering at least one immune checkpoint inhibitor and atleast one retinoid active agent or rexinoid active agent (collectivelyRAR/RXR active agents). In some embodiments, the retinoid active agentis a Retinoic Acid Receptor (RAR) active agent. In some embodiments, therexinoid active agent is a Retinoid X Receptor (RXR) active agent. Insome embodiments, two RAR/RXR active agents are used; they can be twoRAR active agents, two RXR active agents, or a RAR active agent and aRXR active agent. In some embodiments the RAR/RXR active agent acts asan agonist of its receptor while in other embodiments the RAR/RXR activeagent acts as an antagonist of its receptor. In some embodimentsutilizing multiple RAR/RXR active agents, the multiple RAR/RXR activeagents are formulated and administered separately. In some aspects ofthese embodiments, the RAR/RXR active agents are administeredseparately, but during the same treatment session. In other aspects ofthese embodiments, the RAR/RXR active agents are administered indifferent treatment sessions. In other embodiments, the multiple RAR/RXRactive agents are formulated separately, but co-administered (that is,administered during the same treatment session). In still otherembodiments, the multiple RAR/RXR active agents are formulated togetheras a single, common medicament. In many embodiments, the immunecheckpoint inhibitor is an antibody or other polypeptide molecule. Mosttypically such molecules are administered parenterally. In contrast, theRAR/RXR active agents are small molecules that can be administeredorally, for example as pills or capsules and the like. Thus the RAR/RXRactive agents and the immune checkpoint inhibitors may be administeredon independent schedules.

In some embodiments, the immune checkpoint inhibitor interacts with atleast one of CTLA-4, PD-1, TIM-3, LAG-3, PD-L1 ligand, B7-H3, B7-H4,BTLA, ICOS, or OX40. In some embodiments, the immune checkpointinhibitor is an antibody specific for at least one of CTLA-4, PD-1,TIM-3, LAG-3, PD-L1 ligand, B7-H3, B7-H4, BTLA, ICOS, or OX40.

In some embodiments, the retinoid active agent is a RARα antagonist. Insome embodiments, the RARα antagonist is a compound of general formula(I):

wherein R¹, R², R³, and R⁶ are independently H or C₁₋₆ alkyl; R⁴ and R⁵are independently H or F; Ar is phenyl, pyridyl, thienyl, furyl, ornaphthyl; X is C(CH₃)₂, O, S, or NR⁷, wherein R⁷ is H or C₁₋₆ alkyl; X¹is H or halogen such as F, Cl or Br; and R⁸ is H or OH.

In some embodiments, the RARα antagonist is AGN194301, AGN193491,AGN193618, AGN194202, or AGN194574.

In some embodiments, the RARα antagonist is a compound of generalformula (II):

wherein R¹ and R² are independently C₁₋₆ alkyl; X is O, S, or CH₂; Y isO, S, CH₂, or NR³, wherein R³ is C₁₋₆ alkyl; Z is Cl or Br; W is H orOH; and U is independently H or F. In some embodiments, the RARαantagonist is:

In some embodiments, the RARα antagonist is a compound of generalformula (III):

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; Ar isphenyl, pyridyl, thienyl, furyl, or naphthyl; X is O, S, N, or CH₂; W isH or OH; and Z is Cl or Br.

In some embodiments, the RARα antagonist is:

In some embodiments, the RARα antagonist is BMS185411, BMS614,Ro41-5253, or Ro46-5471.

In some embodiments, the retinoid active agent is a RAR agonist. In someembodiments, the RAR agonist is:

In some embodiments, the RAR agonist is a RARγ selective agonist ofgeneral formula (IV):

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; and Xis O, S, CH₂, C(R⁴)₂, or NR⁵, wherein R⁴ and R⁵ are independently H orC₁₋₆ alkyl.

In some embodiments, the RAR agonist is a RARγ selective agonistselected from CD437, CD2325, CD666, LY2813631, and BMS961.

In some embodiments, the retinoid active agent is a RXR antagonist. Insome embodiments, the RXR antagonist is:

In some embodiments, the RXR antagonist is AGN195393, or LGN100849.

In some embodiments, the methods comprise additionally administering atleast one cancer chemotherapy agent.

In some embodiments, the methods comprise administering at least tworetinoid active agents. In some embodiments, the two retinoid activeagents are a RARα antagonist and a RARγ agonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C shows that RAR receptor specific agonists regulate FoxP3,α4β7, and CCR9 expression. Purified CD4⁺ CD25⁻ FoxP3⁻ cells werecultured in media with the specified concentration of each RAR agonistand analyzed by flow cytometry for FoxP3 (FIG. 1A), α4β7 (FIG. 1B), andCCR9 (FIG. 10) expression in total CD4 T cells. FoxP3 results arerepresentative of 3 independent experiments. CCR9 and α4β7 results arerepresentative of multiple experiments.

FIG. 2 shows the extent that compound 5183 activates transcription fromRARα, RARβ, and RARγ using a transactivation assay.

FIG. 3 A-B shows the extent to which the RXR specific agonist, IRX4204promotes the formation of FoxP3+ Treg cells and inhibits the formationof Th 17 cells.

DETAILED DESCRIPTION

Disclosed herein are compounds for potentiation of immune checkpointtargeted therapies. Compounds which act on retinoic acid receptors (RAR)and retinoid X receptors (RXR) potentiate the activity of immunecheckpoint targeted agents. By potentiation it is meant that the immunecheckpoint targeted agent has greater and/or more rapid effect when theRAR/RXR active agent is used with the immune checkpoint targeted agentthan when the RAR/RXR active agent is not used with the immunecheckpoint targeted agent or, similarly, that a given degree of effectcan be obtained with a smaller dosage or concentration of the immunecheckpoint targeted agent when the RAR/RXR active agent is also usedthan would be required if the RAR/RXR active agent were not used.

Retinoic acid (RA), at higher pharmacological concentrations, causesanti-inflammatory effects by increasing levels of suppressive CD4⁺regulatory T cells (Treg cells). RA affects this function by enhancingexpression of the transcription factor Fox P3 which is the masterregulator of Treg cell differentiation. RA also reduces the levels ofpro-inflammatory Th17 cells. RA elicits these effects by activating theRARα subtype of retinoic acid receptors. The above functions of RA orRARα selective agonists results in these compounds contributing toresistance of tumors to immunotherapy. The increased levels ofsuppressor Treg cells impede the anti-tumor activity of the T cellsproduced by immunotherapy. The complement of T cells attacking the tumoris also reduced by the RARα agonist since it reduces the levels of Th17cells. Conversely, an antagonist of RARα sensitizes tumors toimmunotherapy because the RARα antagonist reduces levels of thesuppressive Treg cells and also increase levels of the effector Th17cells. Thus, in one embodiment disclosed herein, a target cancer istreated with a combination of one or more immune checkpoint targetingagents in combination with an RARα antagonist.

In another aspect of RA function, it has been shown that physiologicalconcentrations of RA are critical for the development of T cell mediatedimmune responses. RA signaling to T cells is a critical early mediatorof CD4⁺ T cell effector function. Using T cells expressing dominantnegative RARα (dnRARα), a modified RARα which abrogates RAR function, ora RAR antagonist, it was shown that RA signaling through RARα isrequired for T cell mediated functions such as skin graft rejection.Thus, in the context of cancer immunotherapy, use of RARα antagonists,or RARα inverse agonists, in combination with immune checkpointtargeting agents has counteracting effects: it can promote anti-tumoreffects by decreasing levels of suppressive Treg cells, but suchantagonists can also reduce anti-tumor effects by blocking CD4⁺ T celleffector function. In this context, the use of RARα antagonists incombination with cancer immunotherapy may be of limited value and mayeven be detrimental.

In another embodiment disclosed herein, the RA signaling that iscritical for the development of a T cell mediated immune response ismediated by RARγ. In the above scenario, the sole use of RARαantagonists in conjunction with cancer immunotherapy will result only ina reduction of suppressor Treg cells and consequently in a limitedenhancement of the anti-tumor effects of the immunotherapy. However,that approach does not take advantage of the early effects of RA orother RAR agonists acting through RARγ on promoting CD4⁺ T cell effectorfunction and the potential substantial enhancement of anti-tumor effectsof co-administered cancer immunotherapy. Thus, RAR agonists which actspecifically through RARγ will promote CD4⁺ T cell effector functionwithout increasing Treg cells and such RARγ selective agonists willsubstantially enhance the anti-tumor effects of cancer immunotherapy. Inyet another embodiment, the cancer immunotherapy is used to treat atumor together with a combination of a RARα antagonist and a RARγagonist. In this situation, the retinoid compounds will enhance theanti-tumor activity of the immunotherapy by the following mechanisms:the RARγ agonist will facilitate the development of a robust CD4⁺ T cellmediated immune response released by the immune checkpoint blockingagent; the RARα antagonist will reduce the level of suppressor Tregcells and maintain the level of Th17 cells thereby minimizing modulationof the anti-tumor effects of the immunotherapy.

In another embodiment, RXR antagonists promote the formation ofsuppressor Treg cells and inhibit the formation of effector Th17 cells.The use of a RXR antagonist (or inverse agonist) in combination withcancer immunotherapy will enhance anti-tumor activity by decreasingformation of suppressor Treg cells and by increasing levels of Th17effector cells.

In summary, the following classes of compounds will be useful incombination to increase the anti-tumor activity of cancer immunotherapy:RARα antagonists, RARγ agonists, and RXR antagonists. In the methodsdisclosed herein, one or more immune checkpoint-targeting molecules (forexample, anti-CTLA-4 antibodies, anti-PD1/PD-L1 antibodies) areadministered in combination with one or more of RAR/RXR active agents(for example, RARα antagonists, RARγ agonists, RXR antagonists), with orwithout other agents to treat certain cancers. The properties of RARαantagonism and RARγ agonism maybe present together in the same molecule.Thus, the same molecule acting as an antagonist at RARα can reduce Tregcell formation and, simultaneously, acting as an agonist at RARγ furtherreduce Treg cell formation and promote CD4⁺ T cell effector function. Inthe same manner, the properties of RXR antagonism may be separatelycombined with the properties of RARα antagonism or RARγ agonism indistinct molecules. As used herein, the term “retinoid active agents”encompasses, without limitation, any compound acting on a RAR. Nonlimiting examples of retinoid active agents are RARα antagonists andRARγ agonists. As used herein, the term “rexinoid active agents”encompasses, without limitation, any compound acting on a RXR. A nonlimiting example of a rexinoid active agent is a RXR antagonist.

The term “agonist” as used herein shall be understood to mean a compoundwhich binds to a receptor and activates it, producing gene transcriptionand a subsequent pharmacological response (e.g., contraction,relaxation, secretion, enzyme activation, etc.). As used herein, theterm “RARγ agonist” refers to a compound that binds to RARγ with ahigher affinity compared to binding with another molecule, such as adifferent RAR. In exemplary embodiments, a RARγ agonist is selective forRARγ over RARα and/or RARβ. Thus, a RAR selective agonist tends to bindto a particular RAR receptor target with high binding affinity. As usedherein, the term “agonist” includes selective agonists.

The term “antagonist” as used herein, refers to a compound thatattenuates the effect of an agonist by binding in the same site as anagonist without activating the receptor. An antagonist by itself willnot affect the gene transcriptional activity of the unoccupied receptor.Conventionally, a RARα antagonist is a chemical agent that inhibits theactivity of an RARα agonist. As used herein, the term “RXR antagonist”refers to compounds that bind to RXR and do not activate it, but insteadantagonize transcription produced by a RXR agonist. As used herein, theterm “antagonist” includes selective antagonists.

The term “inverse agonist” as used herein shall be understood to mean acompound which produces an effect opposite to that of an agonist, yetacts at the same receptor. An inverse agonist by itself will reduce thebasal gene transcriptional activity of the unoccupied receptor.

RARα Antagonists

In certain embodiments, the RARα selective antagonist is a compoundrepresented by the general formula (I):

wherein R¹, R², R³, and R⁶ are independently H or C₁₋₆ alkyl; R⁴ and R⁵are independently H or F; Ar is phenyl, pyridyl, thienyl, furyl, ornaphthyl; X is C(CH₃)₂, O, S, or NR⁷, wherein R⁷ is H or C₁₋₆ alkyl; X¹is H or halogen such as F, Cl or Br; and R⁸ is H or OH. Each combinationof R groups and each combination of their independently selectedsubstituents defines a distinct individual embodiment.

An exemplary RARα selective antagonist of the general formula (I) is thecompound AGN194301:

Other exemplary RARα antagonists of the general class of general formula(I) include, but are not limited to, AGN193491, AGN193618, AGN194202,AGN193625, and AGN194574.

In other embodiments, the RARα selective agonist is a member of theclass of compounds represented by general formula (II)

wherein R¹ and R² are independently C₁₋₆ alkyl; X is O, S, or CH₂; Y isO, S, CH₂, or NR³, wherein R³ is C₁₋₆ alkyl; Z is Cl or Br; W is H orOH; and U is independently H or F. Each combination of R groups and eachcombination of their independently selected substituents defines adistinct individual embodiment.

An exemplary RARα selective antagonist of the class represented bygeneral formula (II) for use in the methods disclosed herein isrepresented by the following structure (VTP196696):

In other embodiments, RARα selective antagonists are compounds of thegeneral formula (III).

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; Ar isphenyl, pyridyl, thienyl, furyl, or naphthyl; X is O, S, N, or CH₂; W isH or OH; and Z is Cl or Br. Each combination of R groups and eachcombination of their independently selected substituents defines adistinct individual embodiment.

An exemplary compound of general formula (III) is AGN194777.

Other exemplary RARα antagonists include, but are not limited to,BMS185411, BMS614, Ro41-5253, and Ro46-5471.

Additional RAR antagonists or inverse agonists are described in U.S.Pat. Nos. 6,037,488, 5,612,356, 5,776,699, 5,958,954, 5,877,207,6,225,494, 6,455,701, 5,723,666, 5,739,338, and 5,919,970, and US PatentApplication 2009/0176862, all of which are incorporated by referenceherein for all they disclose of RAR antagonists.

RARγ Agonists

Exemplary RAR agonists are disclosed in U.S. Pat. Nos. 5,234,926,4,326,055, 5,324,840, 5,824,685, and 6,452,032, including but notlimited to the following compounds.

Another exemplary RAR agonist is AGN 190168.

Although compounds such as AGN190183, AGN190205, AGN190168 (tazarotene)are RARγ agonists they are not RARγ selective since they activate RARαand/or RARβ as well. It may be preferable to use RARγ selective agonistssince activation of RARα will inhibit the anti-tumor effects of immunecheckpoint targeted agents by negating the T effector cell activationeffects produced by RARγ activation by increasing production of Tregcells. RARγ selective agonists, on the other hand, will potentiate theanti-tumor effects of cancer immunotherapeutics.

An example of a highly selective RARγ agonist is the compound AGN204647(IRX4647):

Other RARγ selective agonists are members of the family of compounds ofgeneral formula (IV):

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; and Xis O, S, CH₂, C(R⁴)₂, or NR⁵, wherein R⁴ and R⁵ are independently H orC₁₋₆ alkyl. Each combination of R groups and each combination of theirindependently selected substituents defines a distinct individualembodiment.

Additional RARγ selective agonists include, but are not limited to,CD437, CD2325, CD666, LY2813631, and BMS961. Additional RARγ agonistsare described in International Publication WO 02/28810A2 which isincorporated by reference herein for all it discloses regarding RARγagonists.

RXR Antagonists

Exemplary RXR antagonists include, but are not limited to, AGN195393,LGN100849, HX531, LG100754, PA451, PA452, and UVI 3003.

Immune Checkpoint Targeted Cancer Therapeutics

Immune checkpoint therapy targets regulatory pathways in thedifferentiation and activation of T cells to promote the passage of Tcell developmental program through these checkpoints so that anti-tumor(or other therapeutic) activity can be realized. The agents bringingabout immune checkpoint therapy are commonly called immune checkpointinhibitors and it should be understood that it is the check on T celldevelopment that is being inhibited. Thus, while many immune checkpointinhibitors also inhibit the interaction of receptor-ligand pairs (e.g.,anti-PD-1, anti-PD-L1, and CTLA-4), others (such as anti-OX40 andanti-ICOS) act as agonists of targets that release or otherwise inhibitthe check on T cell development, ultimately promoting effector functionand/or inhibiting regulatory function.

Disclosed herein is the use of retinoid and rexinoid receptor activemolecules (RAR/RXR active agents) as potentiators of the anti-tumoreffects of immune checkpoint inhibitor molecules. Molecules whichinhibit immune checkpoint proteins include antibodies which are specificto one or more of PD-1, PD-1 ligand, CTLA-4, TIM-3, LAG-3, B7-H3, andB7-H4.

Programmed death-1 (PD-1) is a checkpoint protein on T cells andnormally acts as a type of “off switch” that helps keep the T cells fromattacking other cells in the body. It does this by binding to programmeddeath ligand-1 (PD-L1), a protein on some normal and cancer cells. WhenPD-1 binds to PD-L1, the T cells will not attack the target cells. Somecancer cells have large amounts of PD-L1, which helps them evade immuneattack. Monoclonal antibodies that target either PD-1 or PD-L1 can boostthe immune response against cancer cells and have shown a great deal ofpromise in treating certain cancers. Examples of monoclonal antibodiesthat target PD-1/PL-L1 include: the anti-PD-1 mAbs nivolumab (OPDIVO®,Bristol-Myers Squibb) and pembrolizumab (KEYTRUDA®, Merck & Co.),BMS-936559 (Bristol-Myers Squibb), pidilizumab (Medivation): and theanti-PD-L1 mAbs durvalumab (MED14736, IMFINZI™, Medimmune), atezolizumab(MPDL3280A; TECENTRIQ®, Hoffman-La Roche), avelumab (BAVENCIO®, EMDSerono). These antibodies have, variously, demonstrated utility intreating a variety of cancers including malignant melanoma (MM), renalcell carcinoma (RCC), Merkel cell carcinoma, urothelial carcinoma, andnon-small cell lung cancer (NSCLC). Non-antibody inhibitors ofPD-1/PD-I1 interaction are also being developed; for example, smallengineered proteins based on stefin A (called AFFIMER® molecules). Inaddition to PD-L1, PD-1 can also bind to PD-L2. In addition to PD-1,PD-L1 can also bind to B7-1 (CD80).

CTLA-4 is an immune checkpoint molecule expressed on the surface of CD4and CD8 T cells and on CD25+, FOXP3+ T regulatory (Treg) cells. CTLA-4generates inhibitory signals that block T cell responses and enablestumor growth. Anti-CTLA-4 mAbs such as ipilimumab (YERVOY®;Bristol-Myers Squibb) cause shrinkage of tumors in animal models.Ipilimumab improves overall survival in MM patients and is approved forthe treatment of MM. Responses have been observed in RCC and NSCLC aswell. Other exemplary anti-CTLA-4 anitbodies include tremelimumab(Medimmune).

The CTLA-4-blocking antibody ipilimumab gives durable responses only ina subset of melanoma patients and its effects on overall survival islimited. This has led to the search for resistance mechanisms to CTLA-4blockade and to the identification of the cytosolic enzyme indoleamine2,3-dioxygenase (IDO) as a potent mediator of melanoma resistance. IDOdirectly suppresses effector T cells and activates suppressive Tregcells thereby modulating the anti-tumor effects of CTLA-4 blockade.Inhibitors of IDO such as 1-methyl-tryptophan have T cell dependentanti-tumor effects and synergize with CTLA-4-blocking antibody tocontrol tumor growth and enhance survival.

TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) is amolecule selectively expressed on IFN-γ-producing CD4⁺ T helper 1 (Th1)and CD8⁺ T cytotoxic 1 (Tc1) T cells. TIM-3 is an immune checkpointreceptor that functions specifically to limit the duration and magnitudeof Th1 and Tc1 T-cell responses. Exemplary antibodies to TIM-3 aredisclosed in U.S. Patent Application Publication 20160075783 which isincorporated by reference herein for all it contains regardinganti-TIM-3 antibodies.

LAG-3 (lymphocyte-activation gene 3; CD223) negatively regulatescellular proliferation, activation, and homeostasis of T cells, in asimilar fashion to CTLA-4 and PD-1 and plays a role in Treg suppressivefunction. Exemplary antibodies to LAG-3 include GSK2831781(GlaxoSmithKline), BMS-986016 (Bristol-Myers Squibb) and the antibodiesdisclosed in U.S. Patent Application Publication 2011/0150892 which isincorporated by reference herein for all it contains regardinganti-LAG-3 antibodies.

The B7 family is a family of costimulatory proteins which are expressedon the surface of antigen-presenting cells and interact with ligands onT cells. B7-H3 (CD276) is one of the molecules in this family. Anantibody to B7-H3, enoblituzumab (EMPLICITI™, Bristol-Myers Squibb) isapproved for treatment of multiple myeloma. Another molecule in thefamily is B7-H4 (V-set domain-containing T-cell activation inhibitor 1),antibodies against which are in development.

Other immune checkpoint inhibitor targets, B- and T-cell attenuator(BTLA), inducible T-cell costimulator (ICOS), OX40 (tumor necrosisfactor receptor superfamily, member 4), and others are potentiallyuseful in the disclosed methods. Several anti-OX40 agonistic monoclonalantibodies are in early phase cancer clinical trials including MED10562and MED16469 (Medimmune), MOXR0916 (Genetech), and PF-04518600 (Pfizer);as is an anti-ICOS agonistic antibody, JTX-2011 (Jounce Therapeutics).

Disclosed herein are methods of potentiating the anti-cancer activity ofimmune checkpoint targeting immunotherapeutics including a CTLA-4inhibitor, a PD-1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, aPD-1 ligand (such as PDL-1), an inhibitor of a PD-1 ligand, an OX40agonist, an ICOS agonist, a B7-H3 protein, an inhibitor of a B7-H3protein, a B7-H4 protein, and an inhibitor of a B7-H4 protein. Incertain embodiments, the inhibitors are antibodies.

The immune checkpoint targeting immunotherapeutic antibodies can bewhole antibodies or antibody fragments. The terms “fragment of anantibody,” “antibody fragment,” and “functional fragment of an antibody”are used interchangeably herein to mean one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Theantibody fragment desirably comprises, for example, one or morecomplementary determining regions (CDRs), the variable region (orportions thereof), the constant region (or portions thereof), orcombinations thereof. Examples of antibody fragments include, but arenot limited to, a Fab fragment, which is a monovalent fragmentconsisting of the V_(L), V_(H), C_(L), and CH₁ domains; a F(ab′)₂fragment, which is a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a single chain Fv, in which the V_(L) and V_(H) domains arejoined by a peptide linker sequence; a Fab′ fragment, which results frombreaking the disulfide bridge of an F(ab′)₂ fragment using mild reducingconditions; a disulfide-stabilized Fv fragment (dsFv); and a domainantibody (dAb), which is an antibody single variable region domain (VHor VL) polypeptide that specifically binds antigen.

In alternative embodiments the antibody is replaced with another proteinthat similarly binds to the immune checkpoint target molecule. In someinstances these non-antibody molecules comprise an extracellular portionof the immune checkpoint target molecule's ligand or binding partner,that is, at least the extracellular portion needed to mediate binding tothe immune checkpoint target molecule. In some embodiments thisextracellular binding portion of the ligand is joined to additionalpolypeptide in a fusion protein. In some embodiments the additionalpolypeptide comprises an Fc or constant region of an antibody.

Methods of Treatment

Provided herein are methods of treating cancer in a mammal byadministering an antibody (or other polypeptide capable of binding) toan immune checkpoint molecule and one or more RAR/RXR active agents.Other embodiments relate to compositions comprising such agents for usein the treatment of cancer and for use in making medicaments for thetreatment of cancer. It is to be understood that the multiple agentsused may be provided in separate compositions or medicaments which maybe administered by separate routes of administration and/or at separatetimes; nonetheless use of such multiple compositions or medicaments iscoordinated so that the patient to whom they are administered receivesthe benefit of the combined, interacting activity the multiple agents.

In some embodiments, the method comprises administering an immunecheckpoint inhibitor and an RAR active agent. In some embodiments, themethod comprises administering an immune checkpoint inhibitor and anRARα antagonist. In some embodiments, the method comprises administeringan immune checkpoint inhibitor and an RARγ agonist. In some embodiments,the method comprises administering an immune checkpoint inhibitor andtwo RAR active agents. In some embodiments, the method comprisesadministering an immune checkpoint inhibitor and an RARα antagonist anRAR agonist. In some embodiments, the method comprises administering animmune checkpoint inhibitor and an RARα antagonist an RARγ selectiveagonist. In certain embodiments, the RARα antagonist is AGN194301,AGN193491, AGN193618, AGN194202, AGN194574, VTP196696, AGN19477,BMS185411, BMS614, Ro41-5253, or Ro46-5471. In some embodiments the RARagonist is AGN190183, AGN190205, AFN204647, or tazarotene. In someembodiments, the RARγ selective agonist is CD437, CD2325, CD666,LY2813631, or BMS961.

In some embodiments, the method comprises administering an immunecheckpoint inhibitor and an RXR active agent. In some embodiments, themethod comprises administering an immune checkpoint inhibitor and an RXRantagonist. In some embodiments, the RXR antagonist is AGN195393 orLGN100849. With respect to the use of multiple RAR/RXR active agents inthe various use or method of treatment embodiments described herein, anyof the disclosed general formula genera, sub-genera thereof, andindividual species may be combined with any other general formulagenera, sub-genera thereof, and individual species, each suchcombination defining an individual embodiment.

The disclosed methods can be used to treat any type of cancer known inthe art, such as, for example, melanoma, renal cell carcinoma, lungcancer, bladder cancer, breast cancer, cervical cancer, colon cancer,gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer,stomach cancer, salivary gland cancer, prostate cancer, pancreaticcancer, or Merkel cell carcinoma. In select embodiments a particulartype of cancer is treated. In other select embodiments a particular typeof cancer is excluded from treatment.

As used herein, the terms “treatment,” “treating,” and the like refer toobtaining a desired pharmacologic and/or physiologic effect. Preferably,the effect is therapeutic, i.e., the effect partially or completelycures a disease and/or adverse symptom attributable to the disease. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. The therapeutically effective amount may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the immune checkpoint inhibitor andone or more retinoid or rexinoid active agents to elicit a desiredresponse in the individual. For example, a therapeutically effectiveamount of a retinoid-active agent of the invention is an amount whichincreases the anti-cancer activity of an immune checkpoint inhibitorantibody or polypeptide.

The term “treating” or “treatment” broadly includes any kind oftreatment activity, including the diagnosis, mitigation, or preventionof disease in man or other animals, or any activity that otherwiseaffects the structure or any function of the body of man or otheranimals. Treatment activity includes the administration of themedicaments, dosage forms, and pharmaceutical compositions describedherein to a patient, especially according to the various methods oftreatment disclosed herein, whether by a healthcare professional, thepatient his/herself, or any other person. Treatment activities includethe orders, instructions, and advice of healthcare professionals such asphysicians, physician's assistants, nurse practitioners, and the likethat are then acted upon by any other person including other healthcareprofessionals or the patient his/herself. In some embodiments, treatmentactivity can also include encouraging, inducing, or mandating that aparticular medicament, or combination thereof, be chosen for treatmentof a condition—and the medicament is actually used—by approvinginsurance coverage for the medicament, denying coverage for analternative medicament, including the medicament on, or excluding analternative medicament, from a drug formulary, or offering a financialincentive to use the medicament, as might be done by an insurancecompany or a pharmacy benefits management company, and the like. In someembodiments, treatment activity can also include encouraging, inducing,or mandating that a particular medicament be chosen for treatment of acondition—and the medicament is actually used—by a policy or practicestandard as might be established by a hospital, clinic, healthmaintenance organization, medical practice or physicians group, and thelike.

A typical dose of an immune checkpoint inhibitor antibody or polypeptidecan be, for example, in the range of 1 pg/kg to 20 mg/kg of animal orhuman body weight; however, doses below or above this exemplary rangeare within the scope of the present disclosure. The daily dose can beabout 0.00001 μg/kg to about 20 mg/kg of total body weight (e.g., about0.001 μg/kg, about 0.1 μg/kg, about 1 μg/kg, about 5 μg/kg, about 10μg/kg, about 100 μg/kg, about 500 μkg, about 1 mg/kg, about 5 mg/kg,about 10 mg/kg, or a range defined by any two of the foregoing values),from about 0.1 μg/kg to about 10 mg/kg of total body weight (e.g., about0.5 μg/kg, about 1 μg/kg, about 50 μg/kg, about 150 μg/kg, about 300μg/kg, about 750 μg/kg, about 1.5 mg/kg, about 5 mg/kg, or a rangedefined by any two of the foregoing values), from about 1 μg/kg to 5mg/kg of total body weight (e.g., about 3 μg/kg, about 15 μg/kg, about75 μg/kg, about 300 μg/kg, about 900 μg/kg, about 2 mg/kg, about 4mg/kg, or a range defined by any two of the foregoing values), and fromabout 0.5 to 15 mg/kg body weight per day (e.g., about 1 mg/kg, about2.5 mg/kg, about 3 mg/kg, about 6 mg/kg, about 9 mg/kg, about 11 mg/kg,about 13 mg/kg, or a range defined by any two of the foregoing values).Therapeutic or prophylactic efficacy can be monitored by periodicassessment of treated patients. For repeated administrations overseveral days or longer, depending on the condition, the treatment can berepeated until a desired suppression of disease or disease symptomsoccurs. However, other dosage regimens may be useful and are within thescope of the invention. The desired dosage can be delivered by a singlebolus administration of the composition, by multiple bolusadministrations of the composition, or by continuous infusionadministration of the composition.

Typical doses of RARα antagonists are 0.01 to 300 mg/m²/day; however,doses below or above this exemplary range are within the scope of thepresent disclosure. The daily dose can be about 0.5 to 100 mg/m²/day, 1to 90 mg/m²/day, 5 to 80 mg/m²/day; or at least 0.02, 0.03, 0.05, 0.07,0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 70 or100 mg/m²/day; or not more than 0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7,10, 15, 20, 25, 30, 50, 60, 70. 80, 90, 100, 125, 150, 175, 200, 225,250, 275, or 300 mg/m²/day; or a range defined by any two of theforegoing values.

Typical doses of RARγ agonists are 0.01 to 300 mg/m²/day; however, dosesbelow or above this exemplary range are within the scope of the presentdisclosure. The daily dose can be about 0.5 to 100 mg/m²/day, 1 to 90mg/m²/day, 5 to 80 mg/m²/day; or at least 0.02, 0.03, 0.05, 0.07, 0.1,0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 70 or 100mg/m²/day; or not more than 0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7, 10,15, 20, 25, 30, 50, 60, 70. 80, 90, 100, 125, 150, 175, 200, 225, 250,275, or 300 mg/m²/day; or a range defined by any two of the foregoingvalues.

Typical doses of RXR antagonists are 0.01 to 300 mg/m²/day; however,doses below or above this exemplary range are within the scope of thepresent disclosure. The daily dose can be about 0.5 to 100 mg/m²/day, 1to 90 mg/m²/day, 5 to 80 mg/m²/day; or at least 0.02, 0.03, 0.05, 0.07,0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7, 10, 15, 20, 25, 30, 50, 70 or100 mg/m²/day; or not more than 0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 5, 7,10, 15, 20, 25, 30, 50, 60, 70. 80, 90, 100, 125, 150, 175, 200, 225,250, 275, or 300 mg/m²/day; or a range defined by any two of theforegoing values.

The average surface area of a human body is generally accepted to be 1.9m² for an adult male, 1.6 m² for an adult female, and 1.33 m² for a12-13 year old child. These values can be used to calculate dose rangesfor daily dosage for the values in the preceding paragraphs. The totaldaily dosage of RAR/RXR active agents can be administered as a singledose or as two doses administered with a 24 hour period spaced 8 to 16,or 10 to 14, hours apart. The RAR/RXR active agents are administered incoordination with the immune checkpoint inhibitor(s) and as abovetherapeutic or prophylactic efficacy can be monitored by periodicassessment of treated patients. For repeated administrations overseveral days or longer, depending on the condition, the treatment can berepeated until a desired suppression of disease or disease symptomsoccurs. However, other dosage regimens may be useful and are within thescope of the invention. The desired dosage can be delivered by a singlebolus administration of the composition, by multiple bolusadministrations of the composition, or by continuous infusionadministration of the composition.

The immune checkpoint inhibitor antibodies and retinoid or rexinoidactive agent can be administered to a mammal using standardadministration techniques, including oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. The checkpoint inhibitorcomposition preferably is suitable for parenteral administration. Theterm “parenteral,” as used herein, includes intravenous, intramuscular,subcutaneous, rectal, vaginal, and intraperitoneal administration. Morepreferably, the composition is administered to a mammal using peripheralsystemic delivery by intravenous, intraperitoneal, or subcutaneousinjection. The retinoid or rexinoid active agent preferably is suitablefor oral administration, for example as a pill, tablet or capsule.

The immune checkpoint inhibitor antibodies and retinoid or rexinoidactive agents disclosed herein of the invention may be administered atsubstantially the same time (within 1 hr of each other) or at differenttimes. In some embodiments, the subject is pre-treated with a retinoidor rexinoid active agent at least 30 min, at least 1 hr, or at least 2hr before administration of the immune checkpoint inhibitor antibody orpolypeptide. In some embodiments, the retinoid or rexinoid active agentis administered after the onset of immune checkpoint inhibitor antibodyor polypeptide administration.

The immune checkpoint inhibitor antibodies and retinoid or rexinoidactive agents disclosed herein may be administered in combination withother drugs, such as at least one other anticancer agent including, forexample, any chemotherapeutic agent known in the art, ionizationradiation, small molecule anticancer agents, cancer vaccines, biologicaltherapies (e.g., other monoclonal antibodies, cancer-killing viruses,gene therapy, and adoptive T-cell transfer), and/or surgery. In otherembodiments the immune checkpoint inhibitor antibodies and retinoid orrexinoid active agents are the only therapeutic reagents administered orthe only treatment given; or the only treatment or reagents given, theprimary utility of which is to promote an anti-cancer immune response;or the only treatment or reagents given, the primary utility of which isto promote a T cell-mediated anti-cancer immune response.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification.

Example 1 RARα Signaling Induces Foxp3 Expression

It is important to determine which of the RAR (RARα, RARβ, RARγ)signaling pathways is important in the induction of Foxp3 expression. Todetermine this, naive CD4⁺ CD25⁻ FoxP3⁻ cells were purified from aFoxp3-GFP mouse using flow cytometry by sorting and isolating based upona GFP⁻ phenotype. These cells were activated polyclonally with αCD3 invitro in the presence of IL-2 and TGF-β. To identify the RAR involved inRA-induced Foxp3 expression, the cultured cells were incubated with RARselective agonists. The cultured cells were then scored for thefrequency of GFP⁺ (Foxp3⁺). Wth respect to the use of selectiveagonists, only the RARα agonist exerted significant impact on theexpression of Foxp3 inducing nearly 100% Foxp3+ T cells, withenhancement on the expression of α4β7 and CCR9 (gut homing receptors)(FIG. 1). The RARγ and RARβ agonists were without effect. These resultsindicate that RARα selective agonists could be useful in reducing asymptom of inflammation or an autoimmune disorder. Conversely, RARαselective antagonists or inverse agonists could be useful todownregulate the production of immunosuppressive Treg cells therebypromoting an immune response, such as an anti-cancer immune response.

Example 2 Compound 5183 is RARα Specific

To determine whether the compounds having a structure of formula I areRARα selective agonists, the compound 5183 was examined for its abilityto bind to RARα, RARβ, and RARγ using a displacement assay to measureagonist binding affinity and a transactivation assay to measure agonistactivity. These results indicate that compound 5183 selectively binds toRARα with high affinity (Table 1) and specifically activates RARα (FIG.2). Such a RARα selective agonist could minimize the adverse effectsrelated to pan-activation including mucocutaneous toxicity, headache,and proinflammatory events in clinical studies.

TABLE 1 5183 Binding Affinities for RARα, RARβ, and RARγ RARα RARβ RARγ4.7 nM >10,000 nM >10,000 nM

Example 3 RARα Selective Agonists Regulates T Cell Differentiation

To determine whether a RARα selective agonist could affect T celldifferentiation, T cells were incubated with a RARα selective agonist todetermine its effect on Foxp3 expression. Naive CD4⁺ CD25⁻ FoxP3⁻ cellswere purified from a Foxp3-GFP mouse using flow cytometry by sorting andisolating based upon a GFP⁻ phenotype. These cells were activatedpolyclonally with αCD3 in vitro in the presence of IL-2 and TGF-β. Thesecells were then cultured in media with various concentrations ofcompound 5183 (a RARα selective agonist) and the expression of FoxP3-GFPwas analyzed by flow cytometry. The RARα selective agonist compound 5183enhanced differentiation of immunosuppressive Treg cells and inhibiteddifferentiation of inflammatory TH17 cells from naïve T cells in vitro(Table 2).

TABLE 2 RARα agonist Effects on T Cell Differentiation Treg cell Th17cell Percent Percent Concen- Differen- Concen- Differen- RARα trationtiation tration tiation agonist (nM) (%) (nM) (%) Compound 0 25 0 325183 0.1 26 0.1 32 1 55 1 21 10 90 10 11 100 ND 100 5

To expand on the finding above, the in vivo effects of a RARα selectiveagonist on T cell differentiation was evaluated in a mouse model. Micewere treated with 100 μg of compound 5183 (a RARα agonist) or anequivalent volume of DMSO (vehicle control) every other day for 10 days.Lympocytes from the blood and spleen were then isolated and FoxP3expression in CD4⁺ T cells was assessed. The data shows that followingadministration of compound 5183 there was a significant increase in thepercentage of Foxp3+ T cells in the spleen and blood of treated mice(Table 3). A two-fold increase in Treg cells should impact significantlyon the overall immunity in the host by reducing immune responses.

TABLE 3 RARα agonist Effects on T Cell Differentiation Foxp3+ Expression(%) Tissue DMSO Compound 5183 Blood 2.4 4.3 Spleen 10 25

Conversely, AGN196996, an RARα selective antagonist, increases Th17 cellnumbers and decreases Treg cell numbers in the above in vitro and invivo assays. Consequently, RARα antagonists increase the immune responseand potentiate the anti-tumor effects of immune checkpoint blockingantibodies.

Example 4 Binding of Test Compounds to RAR and RXR Receptors andActivation of Reporter Genes

Retinoic acid receptor transactivation activity and binding efficienciesare determined essentially as described in U.S. Pat. Nos. 5,298,429 and5,071,773, incorporated by reference herein. Transactivation assaysemploy expression plasmids encoding the full length receptors RARα,RARβ, RARγ, RXRα, RXRβ, and RXRγ. Reporter plasmids containing theherpes virus thymidine kinase promoter and the appropriate retinoic acidreceptor response element (RAREs) or retinoid X receptor responseelement (RXREs) are positioned upstream of an open coding regionencoding firefly luciferase.

Binding assays are performed using a classic competition assay format inwhich cloned receptor RAR and RXR molecules are first loaded with eitherradiolabeled all-trans-retinoic acid (RAR) or radiolabeled 9-cisretinoic acid (RXR), and then the amount of radioactivity liberated withincreasing concentration of test compound is measured.

The assays are used to identify RARα selective antagonists, RARγselective agonists and RXR selective antagonists as disclosed hereinabove.

Example 5 Pharmacological Activation of RARγ Signaling Using RARγAgonists has a Cooperative Effect with Anti-CTLA-4 Antibody in Rejectionof B 16 Melanoma Cells

The anti-tumor effects of anti-CTLA-4 antibody treatment combined with10 nM RARγ agonist (AGN204647 (IRX4647)) are examined in C57BL/6 miceengrafted with B16F10 tumor cells. Mice treated with vehicles only donot show a survival advantage (0%) over untreated control mice. Thesurvival rate of the mice treated with anti-CTLA-4 antibody alone is 40%at 50 days while the mice treated with RARγ agonist alone have a 30%survival in the same time. Remarkably, mice treated with bothanti-CTLA-4 antibody and RARγ agonist have a 100% survival at 50 daysindicating that these two agents cooperate to eliminate the B16 melanomacells. Surviving mice that undergo combination treatment are resistantto re-challenge with twice the dose of live tumor cells indicating theeffective formation of B16-specific memory cells. Importantly, theanti-melanoma effect is obtained with this combination of drugs withoutsigns of acute or delayed toxicity

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used hereinthe terms “about” and “approximately” means within 10 to 15%, preferablywithin 5 to 10%. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof the invention are approximations, the numerical values set forth inthe specific examples are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in their respective testingmeasurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

What is claimed is:
 1. A method of treating cancer comprisingadministering at least one immune checkpoint inhibitor, wherein theimmune checkpoint inhibitor comprises an antibody specific for at leastone of CTLA-4, PD-1, TIM-3, LAG-3, PD-L1, B7-H3, B7-H4, BTLA, ICOS, orOX40, and at least one retinoid active agent wherein the at least oneretinoid active agent comprises a first Retinoic Acid Receptor (RAR)active agent that is a RAR agonist, and wherein the RAR agonist is aRARγ agonist selected from: CD437, CD2325, CD666, LY2813631, BMS961,


2. The method of claim 1, wherein the the at least one retinoid activeagent comprises a RARγ selective agonist selected from CD437, CD2325,CD666, LY2813631, and BMS961.
 3. The method of claim 1, furthercomprising administering a second RAR active agent.
 4. The method ofclaim 3, wherein the second RAR active agent is a RARα selectiveantagonist that is: a compound of general formula (I)

wherein R¹, R², R³, and R⁶ are independently H or C₁₋₆ alkyl; R⁴ and R⁵are independently H or F; Ar is phenyl, pyridyl, thienyl, furyl, ornaphthyl; X is C(CH₃)₂, O, S, or NR⁷, wherein R⁷ is H or C₁₋₆ alkyl; X¹is H or halogen such as F, Cl or Br; and R⁸ is H or OH; (ii) a compoundof general formula (II)

wherein R¹ and R² are independently C₁₋₆ alkyl; X is O, S, or CH₂; Y isO, S, CH₂, or NR³, wherein R³ is C₁₋₆ alkyl; Z is Cl or Br; W is H orOH; and U is independently H or F; (iii) a compound of general formula(III)

wherein R¹ and R² are independently H or C₁₋₆ alkyl; R³ is H or F; Ar isphenyl, pyridyl, thienyl, furyl, or naphthyl; X is O, S, N, or CH₂; W isH or OH; and Z is Cl or Br; or (iv)

BMS185411, BMS614, Ro41-5253, or Ro46-5471.
 5. The method of claim 4,wherein the RARα antagonist is a compound of general formula (I).
 6. Themethod of claim 5, wherein the RARα antagonist is:


7. The method of claim 4, wherein the RARα antagonist is a compound ofgeneral formula (II).
 8. The method of claim 7, wherein the RARαantagonist is:


9. The method of claim 4, wherein the RARα antagonist is a compound ofgeneral formula (III).
 10. The method of claim 4, wherein the RARαantagonist is:

BMS185411, BMS614, Ro41-5253, or Ro46-5471.
 11. The method of claim 1,wherein the method comprises additionally administering at least onecancer chemotherapy agent.
 12. The method of claim 1, wherein thewherein the immune checkpoint inhibitor comprises an antibody specificfor PD-1.
 13. The method of claim 1, wherein the wherein the immunecheckpoint inhibitor comprises an antibody specific for PD-L1.
 14. Themethod of claim 1, wherein the wherein the immune checkpoint inhibitorcomprises an antibody specific for CTLA-4.
 15. The method of claim 1,wherein the wherein the immune checkpoint inhibitor comprises anantibody specific for ICOS.
 16. The method of claim 1, wherein thewherein the immune checkpoint inhibitor comprises an antibody specificfor OX40.
 17. The method of claim 1, wherein the wherein the immunecheckpoint inhibitor comprises an antibody specific for TIM-3.
 18. Themethod of claim 1, wherein the wherein the immune checkpoint inhibitorcomprises an antibody specific for LAG-3.
 19. The method of claim 1,wherein the wherein the immune checkpoint inhibitor comprises anantibody specific for B7-H3 or B7-H4.
 20. The method of claim 1, whereinthe wherein the immune checkpoint inhibitor comprises an antibodyspecific for BTLA.